SECRETION AND PROPERTIES OF THE LARGE AND SMALL LOBES OF THE CHANNEL-FORMING TOXIN AEROLYSIN

Aerolysin is a dimeric protein secreted by Aeromonas spp. that binds t o glycosylphosphatidylinositol-anchored receptors on target cells and becomes insertion competent by oligomerizing. The protein comprises tw o lobes joined by a short arm. The large lobe is thought to be respons ible for channel formation, whereas the small lobe is believed to stab ilize the dimer, and it may also contain the receptor binding site. We cloned and expressed the DNA for both robes of the toxin separately a nd together in A. salmonicida. The large lobe produced alone was secre ted, although more poorly than native protein. The small lobe with the arm produced by itself was not secreted. When the large lobe without the arm was co-produced with the small lobe with the arm, both were se creted, and they co-purified as a stoichiometric complex. Analytical u ltracentrifugation showed that they form a heterotetramer correspondin g to the native dimer. The purified product was nearly as active as ae rolysin, but lost activity and became trypsin sensitive above 25 degre es C. The targe lobe with the arm was also purified. It was shown to b e monomeric, confirming that the small lobe is responsible for dimer s tabilization. The large robe had very low channel-forming activity, al though it was correctly processed by trypsin, and it could form stable oligomers. Surprisingly, the large robe was found to bind to several glycosylphosphatidylinositol-anchored proteins, indicating that it con tains at least part of the receptor-binding domain.